Loss of WHY1 has Specific Effects on Leaf Transcript Abundance
Knockdown of the WHIRLY protein clearly delayed greening of emerging barley leaves (Fig. S2). To determine whether the WHIRLY protein influences other aspects of leaf development, a comparative transcriptome analysis of basal, mid and tip sections of leaves of wild type and two whirly knockdown lines was conducted. Significant differences in transcript abundance (P<0.05) based upon leaf position, genotype or the interaction of the two factors was determined using 2-way analysis of variance which revealed 1732 transcripts dependent on line, 2240 transcripts dependent on leaf region and only 23 which exhibited a distribution in abundance based on an interaction between the two factors (Table S2).
Of the 23 transcripts exhibiting a genotype by leaf region interaction in their patterns of abundance, three transcripts (AK370975, MLOC_56051.1, MLOC_56052.1) that exhibited homology to chlorophyll binding proteins had a low abundance in all sections of wild type leaves and although at higher abundance in W1-1 leaves both genotypes exhibited a pattern of reducing abundance from base to tip. In contrast, W1-7 leaves exhibited a reverse pattern of abundance increasing from base to tip. A similar pattern of abundance was observed for a transcript encoding a thylakoid luminal protein (AK370198). These data are consistent with delayed assembly of the photosystems in WHY-deficient leaves. Two other transcripts encoding proteins with functions in plastid biogenesis and metabolism, respectively also exhibited a genotype by leaf region dependent expression pattern. MLOC_9203.2 encodes a tubulin-like protein with homology to Arabidopsis AT2G36250 encoding an FtsZ protein essential for chloroplast division (Osteryoung, Stokes, Rutherford, Percival, & Lee, 1998) while MLOC_69205.1 encodes an oxaloacetate/malate antiporter acting as a malate valve to balance NADPH/ATP ratios in the plastid (Selinski, & Schiebe, 2019).
We then compared the transcriptome profiles of the three developmental regions in the W1-1 and W1-7 leaves with those of the wild type leaves focusing on the key transcripts discussed above in the wild type developmental pattern (Fig. 4, Table S3). Of these transcripts, most exhibited similar patterns of abundance in the developmental profiles of all genotypes. Notable exceptions were MLOC_70809.1 encoding a homologue of Arabidopsis GATA, NITRATE-INDUCIBLE, CARBON METABOLISM INVOLVED (GNC) transcription factor (AT5G56860) that regulates stomatal development, greening and chloroplast development, MLOC_53744.1 encoding NAC1, a senescence associated transcription factor under the control of auxin, SAG12 (MLOC_47161.1) and two transcripts encoding ARF transcriptions factors (AK364144, MLOC_73144.4) with functions in leaf morphogenesis and development. The patterns of abundance of these transcripts were perturbed in the absence of WHIRLY1 relative to wild type. While SAG12 transcripts were more abundant in all the sections of the W1-7 line than the other genotypes, the ARF and NAC1 transcripts were less abundant in all sections relative to the other genotypes (Fig. 4). These differences are indicative of divergent developmental programmes in leaves deficient in the WHY1 protein.
As a key phenotype of WHY1 knockdown was reduced and delayed greening (Fig. S2) we next sought to examine the shift in abundance of transcripts associated with light signalling and plastid development in leaf regions of different age. Twenty-nine transcripts associated with light dependent plastid biogenesis were identified in the developmental profile of the wild type, of which 22 exhibited a high to low gradient of abundance from the leaf base to leaf tip (Fig. 5). Six transcripts encoded chloroplastic ribosomal proteins and a further six had putative functions in plastid gene expression. Three transcripts (MLOC53063.1, AK3635024 and AK363292) exhibited similarity to AT2G03200 which encodes a putative chloroplast nucleoid DNA binding protein while a further two transcripts (MLOC_65040.1 and MLOC_69013.1) exhibited similarity to AT5G10770 containing similar domain structures (Table S4). The plastid nucleoids comprise multiple copies of DNA, RNA and a range of proteins with functions in replication, gene expression and DNA binding that are believed to control plastid gene expression in a way analogous to chromatin (Powikrowska, Oetke, Jensen, & Krupinska, 2014). A further transcript MLOC_9558.1 encoded a protein with similarity to plastid encoded RNA polymerase alpha subunit, essential for light dependent plastid biogenesis (Yoo et al., 2019). The levels of this transcript declined from base to tip in wild type and WHY 1-1 but the levels of this transcript were high in the base and tip regions of the WHY1-7 leaves.